Prior to describing the background of the invention, it is helpful to define certain terms which are used throughout this specification.
Conjugate: The term "conjugate" applies to any composition that comprises a targeting moiety, such as a biologic response modifier (BRM), (including lymphokines, interferons, erythropoietin, and colony stimulating factors) peptide hormones, antibody or antibody fragment, preferably a monoclonal antibody or monoclonal antibody fragment, and an effector moiety such as a radionuclide, toxin, drug, fibrinolytic enzyme or BRM molecule. The effector moiety can be attached to the targeting moiety either directly or through a linking group, ligand (e.g., bifunctional chelating agent), or a carrier molecule (e.g., albumin, dextran, poly-1-lysine).
Antibody Fragment: An antibody fragment comprises the specific binding regions of the antibody molecule corresponding to the variable and first constant regions on heavy and light chains. Examples of antibody fragments include F(ab')2, Fab' and Fab fragments. An antibody fragment can also include only the variable regions (Fv). Antibody fragments may also include chimetic molecules wherein constant regions of the antibody are substituted with constant regions of immunoglobulin from a different species.
Immunogenicity: Immunogenicity is a measure of the ability of a targeting protein or therapeutic moiety to elicit an immune response (humoral or cellular), when administered to a host. The present invention is concerned with the immunogenicity of the conjugates and their component parts.
Targeting Protein: In this specification a targeting protein is defined as a protein which is capable of binding to a cellular target or other target site, including but not limited to: antibodies and fragments thereof; lymphokines such as IL-1, 2, 3, 4, 5, and 6; .alpha., .beta., and .gamma. interferon; erythropoietin; colony stimulating factors such as G-CSF, GM-CSF and M-CSF; other biologic response modifiers and peptide hormones such as melanocyte stimulating hormone (MSH), follicle stimulating hormone (FSH), leutenizing hormone (LH), and human growth hormone (HGH); fibrinolytic enzymes and serum proteins (e.g., low density lipoproteins).
Serum Half-Life: Serum half-life is a time point at which half of the administered amount of targeting protein or conjugate thereof remains in the serum or plasma. Serum determinations over a series of time points can generate a curve which is useful for determining whole body exposure to an agent.
The advent of recombinant DNA and monoclonal antibody technologies has spawned a renewed interest in proteins that may have pharmaceutical applications. These technologies have made possible, for the first time, the large-scale production of substantially purified, homogeneous proteins with little lot-to-lot variation. Protein molecules such as interleukin-2, tissue plasminogen activator, human insulin, atrial naturetic peptide, interferons, monoclonal antibodies to tumor-associated antigens, and to effector cell subsets and fragments and conjugates thereof, have either received marketing approval in the United States or are currently undergoing multisite clinical trials.
In particular, there has been considerable interest in the "magic bullet" approach to cancer therapeutics using monoclonal antibodies conjugated to drugs, toxins, radionuclides and biological response modifiers. More recent efforts have been devoted to the conjugation of cytotoxic or antineoplastic drugs to specific antibodies, such as monoclonal antibodies, to produce conjugates which can selectively target tumor cells while sparing normal tissues.
A large number of different classes of agents have been considered. These agents include beta- and alpha-emitting isotopes, plant and bacterial toxins, and a variety of antineoplastic drugs, including intercalating agents, antimetabolites, alkylating agents and antibiotics. It is desirable to conjugate chemotherapeutic drugs to targeting molecules such as antibodies to decrease their toxicity directed toward normal tissues.
Conjugates of radioisotopes, drugs and toxins, as well as biologic response modifiers, may be potentially useful in the treatment of cancer and autoimmune diseases, and in the abrogation of transplant rejections. Each class of agent conjugated to an antibody provides conjugates having properties which may be useful for the treatment and diagnoses of cancers and other diseases, but each type also has potential drawbacks.
Radiotherapeutic conjugates, for example, are limited by the low dose of antibody that reaches the tumor and by the sensitivity of the bone marrow to the radiation dose absorbed from the circulating levels of antibody. Toxin conjugates are often potent cytotoxic agents, but suffer from rapid serum clearance usually by the liver. This results in poor tumor delivery. Conjugates formed with cytotoxic drugs are usually of lower potency than toxin conjugates. However, cytotoxic drug conjugates are extracted less rapidly by the liver and reticuloendothelial system (RES), resulting in improved tumor delivery properties.
Similarly, molecules such as tissue plasminogen activator (TPA) and interleukin-2 (IL-2) and some of the interferons have characteristic short serum half-lives. This results in decreased bioavailability for target tissue and, in some instances, increased toxicity. The common problem affecting all four types of conjugates is the limitation of the effective dose of the cytotoxic agent (radionuclide, toxin or drug) attached to the antibody or targeting protein that reaches the tumor site or receptor on potential effector cells.
It has been a goal of therapy and diagnostic imaging with conjugates to increase bioavailability of the circulating conjugate to the tumor site(s). Nonspecific binding of the conjugate to normal cells can occur by nonspecific interactions of the antibody, or agents conjugated to the antibody. Nonspecific binding of the antibody components can occur with cells of the RES tissue. An antibody's Fc region can interact with receptors both for Fc and complement components bound to antibody. One method that has been developed to reduce nonspecific interactions of antibody with RES cells is by treatment of the conjugate with an amphipathic molecule, such as an anionic surfactant, as disclosed in U.S. patent application Ser. No. 767,493, filed Aug. 20, 1985, and now abandoned. The entire disclosure of this patent application is hereby incorporated by reference.
Another method for increasing bioavailability of an conjugate is disclosed in U.S. patent application Ser. No. 917,176, filed Oct. 9, 1986, and now abandoned. The entire disclosure of this patent application is hereby incorporated by reference. As disclosed in U.S. patent application Ser. No. 917,176, an irrelevant antibody or conjugate is pre-injected, thereby swamping the mechanism(s) of nonspecific uptake of the pharmaceutically active conjugate.
Another method whereby nonspecific interactions of the conjugates with the Fc receptor can be minimized is to remove the Fc portion of the antibody with a proteolytic enzyme to create an antibody fragment as the targeting moiety of the conjugate. The use of an antibody fragment as the targeting agent still does not eliminate the problems of nonspecific interactions and binding of the conjugate to normal cells. As an example, in order to increase the sensitivity of radionuclide/antibody imaging, investigators have injected radiolabeled antibodies intralymphatically to detect disease bearing lymph nodes. Despite success in animal models, the method has not been of use in man because of nonspecific accumulation of radiolabel in both diseased and nondiseased lymph nodes. To overcome this nonspecificity, antibody fragments were produced and administered by this route for detection of malignant melanoma. Irrelevant, as well as relevant, Fab antibody fragments showed enhanced accumulation in tumor-involved nodes and less uptake in noninvolved nodes following subcutaneous or intralymphatic administration. (Nelp et al., "Preliminary Studies of Monoclonal Antibody Lymphoscintigraphy in Malignant Melanoma," J. Nucl. Med. 28:34-41, 1987.) Thus, even antibody fragments can undergo nonspecific interactions when administered by the intralymphatic route.
Nonspecific binding to normal cells by the conjugate can be further reduced by understanding the nature of the nonspecific binding. For example, the nonspecific binding of a radiolabeled antibody is increased by the use of radionuclides/chelate systems in which the chelate is not stable enough not to allow leaching of the radiolabel. For example, DTPA, a commonly used chelate for Indium-111 (In-111), does not bind In-111 avidly enough, allowing the leaching (e.g., 10%/day) from the radioconjugate. Free In-111 can then bind to transferrin and then the transferrin binds to receptors in RES tissues. The use of radionuclides can be improved by the use of more stable chelate systems as well as the use of radionuclides that are rapidly excreted upon dissociation from the conjugate.
Conjugates useful for cancer therapeutic purposes will require multiple administrations. Since the targeting protein of the conjugate is a monoclonal antibody or monoclonal antibody fragment of murine (mouse) origin, a host immune response will be generated against the antibody or the agent conjugated to the antibody. Nonspecific uptake into normal tissues, especially RES tissues, through any reception mediated mechanism, would result in catabolism of the conjugate to peptides by macrophages with presentation to immunocompetent T-cells and B-cells. These peptides could then be recognized by T-cell dependent or independent mechanisms and an antibody response generated. Conjugation of toxins or drugs to antibodies, which increase liver accumulation, would also enhance the immunogenicity of both the antibody and the agent conjugated to antibody. Generation of antibodies to components of the conjugate can lead to further nonspecific uptake of circulating conjugate into certain organs, especially RES tissue. Thus, biodistribution, serum clearance, normal organ accumulation, and excretion all impact the degree of immunogenicity of the conjugate and the ability to give multiple administrations to the patient.
Conjugates are not the only proteins useful for targeted imaging or therapy. Lymphokines like IL-2; .alpha., .beta., and .gamma. interferon, all termed "biologic response modifiers (BRMs)"; as well as TNF (tumor necrosis factor); and TPA (tissue plasminogen activator), share common features of requiring in vivo administration to target intra or extravascular receptors. These proteins can be removed from the circulation by either binding to cellular receptors or by excretion/catabolism. It is difficult to estimate what process accounts for the largest amount of protein removed from the circulation, however, modification of the protein to reduce receptor binding is not feasible since that may also reduce its effect or function. Thus, modification of excretion/metabolism may represent the best means for increasing serum half-life of these agents.
This has been recently addressed for TPA, an agent useful for blood clot lysis and imaging. The serum half-life of TPA was increased by producing a mutant protein by recombinant methods which had glycosylation sites and then producing the mutant in eukaryotic cells (Lau et al., Biotechnology 5: 953, 1987). Lau et al. showed that, in addition to increased serum half-life, lower doses of TPA were required to give the same in vivo fibrinolytic activity as non-glycosylated TPA. Glycosylation has been shown previously with many proteins to effect metabolism and excretion.
Thus, the ability to increase the serum half-life of proteins administered in vivo can have substantial impact on the efficacy and therapeutic index of such proteins. Therefore, a method is needed to reduce the nonspecific uptake and excretion of these protein molecules, thereby improving their serum half-lives and thus their availability for target tissues.